Abstract

Gap junctions play critical roles in tissue function and homeostasis. Connexin43 (Cx43) is a major gap junction protein expressed in the mammalian heart and other tissues and may be regulated by its interaction with other cellular proteins. Using the yeast two-hybrid screen, we identified a novel Cx43-interacting protein of 85-kDa, CIP85, which contains a single TBC, SH3, and RUN domain, in addition to a short coiled coil region. Homologues containing this unique combination of domains were found in human, D. melanogaster, and C. elegans. CIP85 mRNA is expressed ubiquitously in mouse and human tissues. In vitro interaction assays and in vivo co-immunoprecipitation experiments confirmed the interaction of endogenous CIP85 with Cx43. In vitro interaction experiments using CIP85 mutants with in-frame deletions of the TBC, SH3, and RUN domains indicated that the SH3 domain of CIP85 is involved in its interaction with Cx43. Conversely, analysis of Cx43 mutants with proline to alanine substitutions in the two proline-rich regions of Cx43 revealed that the P(253)LSP(256) motif is an important determinant of the ability of Cx43 to interact with CIP85. Laser-scanning confocal microscopy showed that CIP85 colocalized with Cx43 at the cell periphery, particularly in areas reminiscent of gap junction plaques. The functional importance of the interaction between CIP85 and Cx43 was suggested by the observation that CIP85 appears to induce the turnover of Cx43 through the lysosomal pathway.

Homologues of CIP85 and their functional domains. Panel A, schematic diagram of CIP85 and a human CIP85 homologue (DJ1042K10.2.1). Both proteins contain TBC, SH3, and RUN domains and coiled coil (C) regions. The region corresponding to CID62 is shown, which encompasses the entire SH3 domain and the NH2-terminal half of the RUN domain of CIP85. The epitope region (Q31-KEESSEQPELCYDE45) used for preparation of the CIP85 antibody is shown. The cDNA probe used to detect the expression of CIP85 mRNA in human tissues by Northern blot analysis is also indicated. Panel B, multiple protein sequence alignment (Clustal W) of TBC domains from CIP85 of mouse (GenBank accession number AY382616), RN-tre of human (GenBank protein accession number NP_055503), GAPCenA of human (GenBank protein accession number NP_036329), and Gyp1p of S. cerevisiae (GenBank protein accession number Q08484). The black shaded areas represent residues matching the consensus sequence, and the gray shaded areas represent residues of conservative substitution. Three conserved “fingerprint” motifs (RXXXW, IXXDXXR, and YXQ) among the members of the Gyp protein family (), in six shared regions named A–F, are shown (). A spade denotes R165 in CIP85 that is conserved, and the corresponding site is critical for Gyp1p and Gyp7p GAP activity (). A diamond denotes R120 in CIP85 that is conserved, and the corresponding site may play a role in the stabilization of the Gyp1p GAP domain (). Panel C, multiple protein sequence alignment (Clustal W) of RUN domains from CIP85, Rabip4 (GenBank protein accession number NP_058039), and RPIP8 (GenBank protein accession number NP_058039) of mouse. Six shared regions named A–F are shown ().

Expression of CIP85 mRNA in mouse and human tissues. Panel A, a [α-32P]-dCTP-labeled CID62 fragment of CIP85 cDNA () was used to probe a membrane containing 2 μg/lane of mRNA derived from various mouse tissues. Panel B, a [α-32P]-dCTP-labeled probe fragment of human CIP85 cDNA () was hybridized to a membrane containing 2 μg/lane of mRNA derived from various human tissues.

Interaction of CIP85 with Cx43 in vitro. Lysates from bacteria expressing Flag-CIP85 or the vector alone were incubated with or without lysates containing HA-Cx43 (lanes 1−3). Flag-CIP85 complexes were collected on glutathione-agarose beads and immunoblotted with a Cx43 antibody. Lysates containing either Flag-CIP85 (lane 4) or HA-Cx43 (lane 5) were used as positive controls.

Interaction between endogenous CIP85 and Cx43 in vivo. Panel A, detection of endogenous CIP85 and Cx43 in 5 mg of lysates from HEK293 cells using the CIP85 or Cx43 antibodies. The CIP85 antibody was incubated without (lanes 1 and 2) or with (lanes 3 and 4) 0.3 μg/μL CIP85 epitope peptide before immunoblotting. Lysates (0.1 mg) of HEK293 transfectants coexpressing exogenous CIP85 and Cx43 were used as positive controls (lanes 2 and 4). Panel B, nontransfected HEK293 cells or HEK293 transfectants expressing exogenous Cx43 were lysed in 300 μL of 0.2% NP-40 lysis buffer. A Flag antibody or the CIP85 antibody was used to immunoprecipitate the protein complexes, and the associated Cx43 was detected with a Cx43 antibody (lanes 2−4). Lysates of HEK293 transfectants coexpressing CIP85 and Cx43 were used as positive controls (lane 1). The increased amount of Cx43 co-immunoprecipitated with CIP85 in HEK293 cells transfected with Cx43 was quantitated by densitometry, and the signal intensity was normalized to that obtained from nontransfected HEK 293 cells for comparison.

Identification of the Cx43-interacting domain in CIP85 by analyses of in-frame deletion mutants. Panel A, schematic diagrams of the CIP85 mutants with in-frame deletions of the TBC domain (ΔTBC), SH3 domain (ΔSH3), or RUN domain (ΔRUN). □: Flag epitope located at the NH2-terminus. Panel B, bacterial lysates containing Flag-CIP85wt (lane 2), the Flag-CIP85ΔTBC (lane 4), Flag-CIP85ΔSH3 (lane 6), or Flag-CIP85ΔRUN (lane 8) mutants were used in the in vitro interaction assays against HA-Cx43. The Flag-CIP85 present in the assays and the associated Cx43 were detected by immunoblotting with Flag or Cx43 antibodies (lanes 2, 4, 6, and 8). Bacterial lysates containing HA-Cx43 (lane 1), Flag-CIP85wt (lane 3), and the various mutants (lanes 5, 7, and 9) were used as positive controls to show the migration positions of these proteins. The signal intensities quantitated by densitometry were normalized for comparison. Panel C, affinity gel-purified Flag-CIP85wt (lane 2) and the Flag-CIP85ΔSH3 mutant (lane 4) were used in the in vitro interaction assays against HA-Cx43. The Flag-CIP85 present in the assays and the associated Cx43 were detected by immunoblotting with Flag or Cx43 antibodies (lanes 2 and 4). Bacterial lysates containing HA-Cx43 (lane 1), Flag-CIP85wt (lane 3), and Flag-CIP85ΔSH3 (lane 5) were used as positive controls.

Identification of the CIP85-interacting region in Cx43 by analyses of proline site substitution mutants. Panel A, schematic diagrams of the Cx43 proline to alanine substitution mutants (Cx43NPA and Cx43CPA). The primary structure of Cx43 includes four transmembrane regions (TM1−4) indicated by black shaded boxes and two proline-rich regions (P253LSP256 and P274TAPLSPMSPP284) indicated by the open boxes. Panel B, HEK293 cells transiently coexpressing Flag-CIP85 along with either the Cx43NPA mutant or the Cx43CPA mutant were lysed in 300 μL of 0.2% NP-40 lysis buffer. A Flag antibody was used to immunoprecipitate subpopulations of the protein complexes, and the associated Cx43 was detected by immunoblotting with a Cx43 antibody (lanes 1 and 3). Five microliters of lysates of HEK293 transfectants were used as positive controls (lanes 2 and 4).

Subcellular colocalization of Cx43 and CIP85. Panel A, laser-scanning confocal microscopy was performed to determine the subcellular localization of Cx43 and endogenous CIP85 in Hela cells (clone C4) utilizing TRITC-conjugated or FITC-conjugated secondary antibodies. The white arrows indicate regions of colocalization of CIP85 and Cx43. The boxed area in the merged image was magnified to show the regional colocalization of Cx43 and CIP85 in a gap junction plaque. Panel B, HEK293 cells were transiently transfected with Cx43 and Flag-CIP85. Laser-scanning confocal microscopy was performed to determine the subcellular localization of Cx43 and CIP85 with TRITC-conjugated or FITC-conjugated secondary antibodies. The white arrows indicate regions of colocalization of CIP85 and Cx43. Right panel section: an orthoimage showing the colocalization of CIP85 and Cx43 in a gap junction plaque in the X-, Y-, and Z-axes.